Introduction
Bacterial diseases represent a major constraint for Mediterranean aquaculture, particularly in European sea bass (Dicentrarchus labrax), where increased farming intensity and environmental changes are contributing to the emergence of pathogenic species (Reverte et al., 2020). Among these, Aeromonas veronii has been widely recognized as an emerging pathogen associated with disease outbreaks in European sea bass, particularly under elevated temperature conditions (Tanrikul & Dinçtürk, 2021). Recent efforts have therefore focused on the development of reproducible infection models for this species, enabling the evaluation of preventive strategies under controlled conditions. In this context, the incorporation of multiple isolates from different outbreaks has become essential, as intraspecific variability in virulence has been reported for Aeromonas spp., potentially affecting disease outcomes and model reproducibility (Smyrli et al., 2019). Accordingly, this study expands previous work by including new A. veronii strains isolated from European sea bass in different geographical and epidemiological contexts, allowing a broader assessment of pathogenicity. In parallel, Lactococcus garvieae has emerged as a relevant pathogen in marine aquaculture, with recent outbreaks reported in European sea bass farms in Mediterranean countries. This pathogen is associated with septicemia and high mortality rates, particularly under stressful environmental conditions such as elevated temperature and fluctuating salinity (Fouz et al., 2025). Despite its growing importance, infection models under realistic production conditions remain limited.
Therefore, the present study aims to (i) refine and expand infection models for A. veronii using multiple isolates and (ii) develop and validate infection models for L. garvieae under both controlled (Recirculating Aquaculture Systems - RAS) and semi-natural (estuarine pond) conditions representative of Mediterranean aquaculture systems where the effects of climate change are more evident.
Materials and methods
Bacterial isolates of A.veronii and L.garvieae were obtained from diseased European sea bass during field outbreaks and identified using MALDI-TOF and qPCR. Strains were cryopreserved and incorporated into the CTAQUA collection.bExperimental challenges were conducted using European sea bass at pre-ongrowing and ongrowing stages. For A. veronii, multiple isolates were tested using intraperitoneal (IP) injection and bath immersion to evaluate dose-response relationships and inter-strain variability. For L. garvieae, infections by intraperitoneal (IP) injection were conducted under different experimental conditions. On the one hand, trials were performed under standard farming conditions representative of current production situation for this species and pathogens. On the other hand, additional trials were carried out under more extreme environmental conditions, including elevated temperature and salinity fluctuations, designed to simulate those typically observed in estuarine pond systems (esteros) in southern Spain, which are increasingly influenced by climate change. This approach allowed the evaluation of pathogen behavior under both current farming conditions and climate change-driven stress scenarios.
For bath infections, the water level in the system tanks was reduced or 10 L buckets were used, depending on fish size. Under these conditions, fish were exposed to the pathogen for 1 hour at the different tested concentrations, while aeration and animal welfare were continuously monitored. For IP injection challenges, 0.1 mL of bacterial suspension per fish was administered at the desired concentration. Prior to injection, fish were deeply anesthetized using benzocaine. All trials included control groups (no exposed to the pathogen during bath infections or inoculated with Phosphate Buffered Saline (PBS) during IP injection infections) and were performed in triplicate. Mortality, culture parameters, clinical signs, and bacterial re-isolation were recorded.
Results and discussion
For A. veronii, dose-response relationships were consistent across the different isolates, although mortality rates differed significantly among strains (Smyrli et al., 2019). The results confirm that A. veronii isolates exhibit variable virulence depending on virulence factors of the strain, the route of administration, but also fish origin, size and batch, highlighting the importance of incorporating multiple strains and conditions for the development of robust infection models that allow the evaluation of different preventive measures or more versatile and universal treatments, capable of covering the pathogenicity of different strains. L. garvieae induced a septicemic disease in European sea bass, with infection dynamics strongly influenced by environmental conditions (Salogni et al., 2024). Higher temperatures and salinity fluctuations, simulating estuarine system conditions, affected both mortality and disease progression. Comparative analyses between standard and extreme conditions revealed more heterogeneous and severe outcomes under semi-natural conditions. Overall, these findings demonstrate the need to integrate environmental variability into infection models to better reflect real farming conditions and improve the evaluation of preventive strategies.
References
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